Throughout this application various publications are referenced by the names of the authors and the year of the publication within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The synthesis of RNA in vitro by Q.beta. replicase (Haruna & Spiegelman, 1965a) is remarkable because a small number of template strands can initiate the synthesis of a large number of product strands (Haruna & Spiegelman, 1965b). A 100,000-fold increase in RNA can occur during a ten-minute reaction (Kramer et al., 1974). This striking amplification is the consequence of an autocatalytic reaction mechanism (Spiegelman et al., 1968; Weissmann et al., 1968). Single-stranded RNAs serve as templates for the synthesis of complementary single-stranded products. Both the product strand and the template strand are released from the replication complex and are free to serve as templates in subsequent rounds of synthesis (Dobkin et al., 1979). Consequently, the number of RNA strands increases exponentially as the reaction proceeds.
Many investigators have attempted to exploit the auto-catalytic nature of Q.beta. replicase reactions in order to synthesize large amounts of any RNA in vitro. However, Q.beta. replicase does not copy most RNAs. Like other viral RNA-directed RNA polymerases it is highly selective for its own template (Haruna & Spiegelman 1965c). In vivo this enables Q.beta. replicase to distinguish bacteriophage Q.beta. RNA from the vast number of different RNA molecules that are present in Escherichia coli. This template specificity is a consequence of two separate interactions that occur between the replicase and Q.beta. RNA. First, the replicase binds strongly to a unique internal recognition sequence (Weber et al., 1974; Vollenweider et al., 1976; Meyer et al., 1981). Then, product strand synthesis is initiated at a cytidine-rich sequence located at the 3' end of the template (Rensing & August, 1969; Schwyzer et al., 1972). Each of these sequences must be present in both complementary strands for autocatalytic synthesis to occur. A number of strategies have been devised to circumvent these barriers to the synthesis of heterologous RNAs by Q.beta. replicase. Manganese was used to decrease the stringency of the interactions between the replicase and the template (Palmenberg & Kaesberg, 1974; Obinata et al., 1975); primers were used to bypass the normal initiation step (Feix & Hake, 1975; Feix, 1976; Vournakis et al., 1976); and polycytidine tails were added to templates to mimic the required 3'-terminal sequence (Kuppers & Sumper, 1975; Owens & Diener, 1977). These strategies were tried with a wide range of heterologous templates, including rRNAs, viral RNAs and eukaryotic mRNAs. In all cases, the amount of RNA synthesized never exceeded the original amount of template RNA and the products consisted only of complementary strands. Consequently, synthesis was not auto-catalytic and these methods could not approach the efficiency with which Q.beta. RNA is synthesized by Q.beta. replicase.
In a different strategy a poly (A) molecule was inserted between two Q.beta. RNA molecules which had been partially degraded, one from the 3'-end and another from the 5' end. E. coli HrH were infected with these RNA molecules and protoplasts of two phage clones carrying poly (A) in their RNA were obtained after reproduction of the phage in vivo. (Tongjian and Meiyan, 1982). The yield of recombinant RNA molecules produced by this method was very low and its infectivity was between 1/1000 and 1/10000 that of the wild type RNA.
This invention concerns a method for the autocatalytic synthesis of heterologous RNAs in vitro by Q.beta. replicase. Our approach was to construct a recombinant RNA by inserting a heterologous sequence into a natural Q.beta. replicase template. The template we used, MDV-1 (+) RNA (Kacian et al., 1972), is only 221 nucleotides long, and its complete nucleotide sequence (Mills et al., 1973; Kramer & Mills, 1978) and secondary structure (Mills et al., 1980) have been determined. The mechanism of its replication by Q.beta. replicase has been studied in detail (Mills et al., 1978; Dobkin et al., 1979; Kramer & Mills, 1981; Bausch et al., 1983) and has been shown to be fundamentally similar to the replication of Q.beta. RNA. In particular, it possesses a highly structured internal binding site for Q.beta. replicase (Nishihara et al., 1983) and a cytidine-rich 3'-terminal sequence that is required for product strand initiation (Mills et al., 1980). We selected an insertion site at a position where the heterologous sequence would not interfere with these functional regions and where it would not disturb the structure of the MDV-1 RNA. We hoped that the replicase would respond to the recombinant RNA as it would to a natural template.
The recombinant RNA was constructed by cleaving MDV-1 (+) RNA at the selected site and then inserting decaadenylic acid in that site with the aid of bacteriophage T4 RNA ligase. This recombinant RNA was then used as a template in a reaction containing Q.beta. replicase. The product consisted of full-length copies of the recombinant RNA. Furthermore, both complementary strands were synthesized. The reaction proceeded autocatalytically, resulting in an exponential increase in the amount of recombinant RNA.